Electron microscopic tomography is being used at the Albany national biotechnology resource center to determine the internal organization of mitochondria and the interactions of these organelles with other cellular components. There are two aspects of this ongoing work on which they plan to collaborate with Dr. Loew, using the three-dimensional structural information from tomography as input into the "Virtual Cell" program. The project hypothesizes, based on 3-D electron tomographic reconstructions that suggest that mitochondrial cristae are connected by 30-40 nm diameter tubes, that the chemosmotic theory of mitochondrial ATP production may need to be modified to incorporate putative PH gradients imposed by the cristal connections. EM reconstructions of mitochondrial structure will be incorporated into the Virtual Cell to explore the possibility of PH gradients and the attendant spatial gradients of other species including ATP. A second aim of this project is to supply EM-level detai l of the distribution of mitochondrial and ER distributions to the calcium dynamics project in the set of core projects proposed. The investigator will use "Virtual Cell" to test the hypothesis that dynamic gradients of protons (and other ions and metabolites) may exist between the intracristal compartments of mitochondria and the intermembrane or cytosolic compartments. With "Virtual Cell," it should be possible to determine how gradients vary with such parameters as proton pumping rates and bulk diffusional coefficients. Methods of testing the predictions of this method are under development, e.g., targetting of pH-dependent probes (such as GFP-fusion proteins) to internal mitochondrial compartments to detect possible acidification. Preliminary simulations have now been performed based on an experimental 30 cristal image that was supplied to the UCHC group. The results of the initial simulation is displayed on the CBIT website. There is growing evidence that mitochondria take part in cellular Ca++ regulation, e.g., propagation of IP3-mediated Ca++ waves. They are generating 3D reconstructions of regions of cells (liver and oligodendrocytes) showing the spatial relationships between mitochondria and the endoplasmic reticulum, including regions of close apposition between mitochondrial outer membrane and endoplasmic reticulum. In this collaborative project, this structural information will be made available to Dr. Loew for collaborative studies modeling intracellular Ca++ dynamics and mitochondrial Ca++-induced Ca++ release using "Virtual Cell."